The precise regulation by glutamate transporters of glutamate concentrations in and around excitatory synapses is critical for the normal function of excitatory synapses. During cerebral ischemic injury, the third leading cause of death of adults in the United States, extracellular glutamate concentration rises, leading to excitotoxicity caused by excess activation of glutamate receptors. The long-term goal of this research is to understand how glutamate transporters regulate synaptic and perisynaptic glutamate concentrations normally, and how ischemia disrupts glutamate homeostasis to produce excitotoxicity. Although glutamate transporters are well known to be expressed in astrocytes, the identity of the glutamate transporter expressed in the presynaptic terminal of excitatory synapses was unknown, representing a major gap in our knowledge and understanding of excitatory synapses. We discovered that GLT1, previously thought to be exclusively expressed in astrocytes in the mature brain, is expressed in axon terminals in the hippocampus. A major hypothesis of this proposal is that GLT1 is the major glutamate transporter expressed in excitatory terminals throughout the forebrain. The function of GLT1 expressed in axon terminals as opposed to GLT1 expressed in astrocytes is unclear. We hypothesize that expression of GLT1 in neurons is important for the normal function of excitatory synapses, by preventing spillover onto perisynaptic glutamate receptors as well as cross-talk between excitatory synapses. We further hypothesize that the release of glutamate from excitatory terminals is important in the pathogenesis of excitotoxic injury in ischemia. We have generated a mutant mouse in which a critical exon in the GLT1 gene has been flanked with loxP sites, allowing the use of Cre-loxP recombination technology to produce cell-type specific knockouts. In this project we will compare the effects of deletion of GLT1 in astrocytes or in neurons, on glutamate homeostasis and synaptic function under normal conditions and in models of excitotoxic injury. The specific aims are to: 1) Perform phenotypical, morphological, and physiological analysis of mouse lines in which GLT1 is deleted in astrocytes or neurons. 2) Characterize the role of GLT1 expressed in different cell types in the pathogenesis of ischemic injury. 3) Determine whether GLT1 is expressed in excitatory terminals in regions outside the hippocampus. The expression of GLT1 in excitatory terminals has important implications for our understanding of the physiology of excitatory synaptic transmission, synaptic plasticity, and ischemic injury. Using mouse lines that will be produced for this project, we hope to gain important insights into the role of neuronal expression of GLT1 into the normal and abnormal regulation of glutamate at the excitatory synapse. PUBLIC HEALTH RELEVANCE: The long-term goal of this research is to understand how glutamate transporters normally regulate glutamate concentrations at and around the excitatory synapse, and how ischemia disrupts this regulation to produce excitotoxicity-death of neurons caused by excess release of glutamate and activation of glutamate receptors. We discovered that the glutamate transporter GLT1 is expressed in excitatory axon terminals, but the function of glutamate transporters in excitatory terminals, as opposed to astrocytes, is unclear. In this project we make mouse lines in which GLT1 expression is deleted in neurons or in astrocytes to compare the effects of expression of GLT1 in these different locations on glutamate homeostasis and synaptic function under normal conditions and in models of excitotoxic injury.